PROJECT SUMMARY The choroid plexus (ChP) is a vital tissue located in each ventricle in the brain. The ChP is composed of two parallel sheets of epithelial cells with an intervening network of primarily non-neural cell types and vasculature. The ChP (1) produces cerebrospinal fluid (CSF) containing growth-promoting factors for the brain, (2) forms a blood-CSF barrier that gates communication between the central nervous system (CNS) and the systemic milieu, (3) provides for immune cell entry into the brain, and (4) offers an enticing framework for enhanced drug delivery. While ideally positioned to regulate brain function broadly, compared to other neural and non-neural brain systems, the ChP network is surprisingly poorly understood. Progress in understanding its role has been hindered in large part due to the lack of available tools for selectively accessing and controlling the ChP in vivo. Here, we develop a toolkit to enable a modern, system-level approach to ChP study in vivo. We recently used single cell sequencing to comprehensively classify and characterize the resident cells of the ChP. We will use this new map to engineer mouse driver lines expressing Cre- or Flpe-recombinase in specific ChP cell types, allowing precise monitoring and control of each type (Aim I). In Aims II and III, we focus on developing tools to observe and control calcium activity patterns. In Aim II we will develop detailed methods for 3D 2- photon imaging of ChP epithelial cells, including 3D non-rigid alignment of imaging volumes and methods for tracking the same cells across hours, days and weeks. In this Aim, we will also optimize the acquisition and analysis of 2-photon imaging datasets to visualize motility dynamics of ChP immune cells. Our Preliminary Data reveal rich dynamics of both kinds in different ChP cell types. Testing the functional role of calcium activity is greatly aided by the ability to directly manipulate this crucial molecule. In Aim III, we will develop optimal and detailed protocols for optogenetic and chemogenetic regulation of the ChP, initially focusing on the induction of calcium dynamics in epithelial cells. We will actively employ best practices in Open Science, to ensure rapid dissemination of all products of this work, including through a database-integrated website to facilitate easy community access. This toolkit development builds on the ongoing collaboration led by PI Dr. Maria Lehtinen, an expert in ChP function. Drs. Mark Andermann and Christopher Moore are expert at in vivo systems neuroscience, specifically the use and novel development of 2-photon, optogenetic and chemogenetic tools. This successful partnership has produced the Preliminary Data shown throughout.